Various scientific articles, patents and other publications are referred to throughout the specification. Each of these publications is incorporated by reference herein in its entirety.
Apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multi-cellular organisms. Alterations in apoptotic pathways have been implicated in many types of human pathologies, including developmental disorders, cancer, autoimmune diseases, as well as neuro-degenerative disorders.
Thus, the programmed cell death pathways have become attractive targets for development of therapeutic agents. In particular, since it is conceptually easier to kill than to sustain cells, attention has been focused on anti-cancer therapies using pro-apoptotic agents such as conventional radiation and chemo-therapy. These treatments are generally believed to trigger activation of the mitochondria-mediated apoptotic pathways. However, these therapies lack molecular specificity, and more specific molecular targets are needed.
Apoptosis is executed primarily by activated caspases, a family of cysteine proteases with aspartate specificity in their substrates. Caspases are produced in cells as catalytically inactive zymogens and must be proteolytically processed to become active proteases during apoptosis. In normal surviving cells that have not received an apoptotic stimulus, most caspases remain inactive. Even if some caspases are aberrantly activated, their proteolytic activity can be fully inhibited by a family of evolutionarily conserved proteins called IAPs (inhibitors of apoptosis proteins) (Deveraux & Reed, Genes Dev. 13: 239-252, 1999). Each of the IAPs contains 1-3 copies of the so-called BIR (baculoviral IAP repeat) domain and directly interacts with and inhibits the enzymatic activity of mature caspases. Several distinct mammalian IAPs including XIAP, survivin, and Livin/ML-IAP (Kasof & Gomes, J. Biol. Chem. 276: 3238-3246, 2001; Vucic et al. Curr. Biol. 10:1359-1366, 2000; Ashhab et al. FEBS Lett. 495: 56-60, 2001), have been identified, and they all exhibit anti-apoptotic activity in cell culture (Deveraux & Reed, 1999, supra). As IAPs are expressed in most cancer cells, they may directly contribute to tumor progression and subsequent resistance to drug treatment.
In normal cells signaled to undergo apoptosis, however, the IAP-mediated inhibitory effect must be removed, a process at least in part performed by a mitochondrial protein named Smac (second mitochondria-derived activator of caspases; Du et al. Cell 102: 33-42, 2000) or DIABOLO (direct LIP binding protein with low pI; Verhagen et al. Cell 102: 43-53, 2000). Smac, synthesized in the cytoplasm, is targeted to the inter-membrane space of mitochondria. Upon apoptotic stimuli, Smac is released from mitochondria back into the cytosol, together with cytochrome c. Whereas cytochrome c induces multimerization of Apaf-1 to activate procaspase-9 and -3, Smac eliminates the inhibitory effect of multiple LPs. Smac interacts with all IAPs that have been examined to date, including XLIP, c-IAP1, c-IAP2, and survivin (Du et al., 2000, supra; Verhagen et al., 2000, supra). Thus, Smac appears to be a master regulator of apoptosis in mammals.
Smac is synthesized as a precursor molecule of 239 amino acids; the N-terminal 55 residues serve as the mitochondria targeting sequence that is removed after import (Du et al., 2000, supra). The mature form of Smac contains 184 amino acids and behaves as an oligomer in solution (Du et al., 2000, supra). Smac and various fragments thereof have been proposed for use as targets for identification of therapeutic agents. U.S. Pat. No. 6,110,691 to Wang et al. describes the Smac polypeptide and fragments ranging from at least 8 amino acid residues in length. However, the patent neither discloses nor teaches a structural basis for choosing a particular peptide fragment of Smac for use as a therapeutic agent or target.
Similar to mammals, flies contain two IAPs, DIAP1 and DIAP2, that bind and inactivate several Drosophila caspases (Hay, Cell Death Differ. 7: 1045-1056, 2000). DIAP1 contains two BIR domains; the second BIR domain (BIR2) is necessary and sufficient to block cell death in many contexts. In Drosophila cells, the anti-death function of DIAP1 is removed by three pro-apoptotic proteins, Hid, Grim, and Reaper, which physically interact with the BIR2 domain of DIAP1 and remove its inhibitory effect on caspases. Thus Hid, Grim, and Reaper represent the functional homologs of the mammalian protein Smac. However, except for their N-terminal 10 residues, Hid, Grim, and Reaper share no sequence homology with one another, and there is no apparent homology between the three Drosophila proteins and Smac.
In commonly-owned co-pending U.S. application Ser. No. 09/965,967, it is disclosed that the above described biological activity of Smac is related to binding of its N-terminal four residues to a featured surface groove in a portion of XIAP referred to as the BIR3 domain. This binding prevents XIAP from exerting its apoptosis-suppressing function in the cell. It was further disclosed that N-terminal tetrapeptides from IAP binding proteins of the Drosophila pro-apoptotic proteins Hid, Grim and Veto function in the same manner.
Commonly-owned co-pending International Application No. PCT/US02/17342 describes assays for use in high throughput screening or rational drug design of agents that can, like the Smac tetrapeptide or its homologs in other species, bind to a BIR domain of an IAP, thereby relieving IAP-mediated suppression of apoptosis. Those assays are based on competitive displacement of a labeled IAP-binding tetrapetide by a test compound. That application also describes a library of peptides and N-methyl peptide analogs that were demonstrated by the assay method to bind to the BIR3 domain of XIAP.
The use of peptides for in vivo administration as diagnostic or therapeutic agents is associated with certain disadvantages. These include short half-life due to proteolytic degradation in the body, low absorption through intestinal walls and potential immunogenic reactions, as well as expense involved in peptide synthesis. For these reasons, many current efforts in drug development focus on non-peptidic mimetics that mimic the structure and biological activity of bioactive peptides, but possess improved pharmacologic properties and are easier or less expensive to synthesize.
In connection with the Smac tetrapeptides and homologs described above, then, it would be a significant advance in the art to develop partial peptide or non-peptide mimetics of those molecules. Such mimetics should possess the IAP-binding and apoptosis-promoting bioactivity of the Smac peptides, while also having the improved properties associated with non-peptide mimetics, for use as diagnostic and therapeutic agents in the treatment of cancer.